Overview

The central focus of the Section on Nutritional Neurochemistry is to examine the impact of deficiencies in long chain omega-3 fatty acids on adverse neurodevelopmental and psychiatric outcomes. This section translates basic biochemical and biophysical findings into clinical applications. The portfolio of projects include studies in genetic epidemiology, large-scale longitudinal cohort studies, studies of basic metabolism, the development of high throughput high accuracy analytical biochemical methods suitable for population level examinations and randomized placebo-controlled clinical intervention trials among populations potentially affected by insufficient intakes. Active clinical intervention protocols include aggressive subjects with alcohol use disorders, subjects with a history of suicide attempts, pediatric populations with bipolar disorders and disruptive behaviors, and women with severe depressive symptoms in pregnancy. Several military applications are being developed, including compositional analysis of foods currently provided in combat theaters, assessment of increased risk of suicide and Post Traumatic Stress Disorder resulting from inadequate tissue compositions of long chain polyunsaturates and the development of a large-scale prevention trial utilizing omega-3 fatty acids to prevent the development of severe depression, psychiatric distress and adverse outcomes from dramatic brain injury for military personnel in combat theaters.

The first in vivo studies of the essential fatty acid (EFA) metabolism in alcoholics and smokers have been performed within the Section of Nutritional Neuroscience. In addition, our research with infants and adults report some of the first basic studies for normative physiology. Highly sensitive and selective methodology has been developed within our research group allowing for the safe and non-invasive assessment of EFA metabolism in vivo. This approach takes advantage of the stable isotope labeled EFAs, deuterated linoleic and linolenic acids, along with negative, chemical ionization gas chromatography /mass spectrometry to simultaneously measure the precursor and product fatty acids in the blood stream after metabolism and export in the liver. This methodology has advanced with new capabilities to simultaneously measure different components of the EFA pathway and to model the resulting data so as to obtain rate related parameters. Thus, arguably the worldwide lead in the metabolomics of essential fatty acids has been gained.

Our metabolic studies were among the first clinical studies performed in human adults and showed that a diet low in n-3 fats leads to an increase in formation and transport of long chain polyunsaturates to the plasma compartment. Smokers also have somewhat increased activity in this regard. Smoking alcoholics have a further marked increase in the deuterium enrichment of plasma DHA when deuterated alpha-linolenate is orally ingested. Both smoking and alcohol are peroxidative challenges that are hypothesized to lead to increased EFA degradation. The apparently greater level of formation/transport of the fatty acids may, in part, compensate for these losses. When the intensity of the alcohol challenge is too great with respect to its frequency and dosage, metabolism cannot keep pace with the increased catabolism and tissue levels fall, consistent with our findings in many species. This fall in long chain polyunsaturate levels then has adverse consequences for organ function and may lead, in particular, to liver and brain pathology. Dietary impact of these fats is variable in the alcoholic population and may be protective for alcohol-induced organ pathology; this may help to explain why some alcoholics develop liver disease or organic brain syndrome and others do not.

In human infants in the first week of life, our results indicate a general capacity to perform 18- carbon EFA metabolism to 20 and 22-carbon end products for both the n-3 and n-6 pathways. There was a surprising inverse relationship between gestational age and metabolic capacity that was strongest for docosahexaenoic acid (DHA). This is consistent with the rapid formation of neural membranes for which DHA is an important structural constituent during the brain growth spurt in the third trimester of human development. The rate of conversion in these infants is slow and appears inadequate to support the amount of DHA required for optimal brain and other organ development. We concluded, therefore, that infant formula must have preformed DHA added to support optimal nervous system growth and function; formulas in North America have only recently become available with DHA supplements. A requirement for preformed DHA was supported by studies of rat brain development with low n-3 fatty acid content in the diet. It was demonstrated that docosapentaenoic acid (22:5n6) was unable to completely replace brain DHA in this case, especially in rapidly growing brain areas like the cerebellum as well as in the retina. This effect was maximized during rapid brain growth at 10-20 postnatal days.

Projects

1. Adverse neurodevelopmental outcomes associated with inadequate seafood consumption during pregnancy (Hibbeln et al. 2008).
In 2004, the Environmental Protection Agency and FDA advised limiting seafood intake during pregnancy in order to reduce fetal exposure to methyl-mercury (MeHg). However, compliance (<340 g/w) also restricts intake of nutrients essential for optimal neuronal development, in particular DHA. Deficiencies during early development can potentially increase risk of childhood pathological outcomes in verbal functioning and disruptive behavioral problems. Here the study population was mother and infant pairs (n=1 1,875) enrolled in the UK Avon Longitudinal Study of Parents and Children (ALSPAC). This cohort is a representative sample (>85%) of pregnancies in the Avon region near Bristol, UK. Mothers completed a food frequency questionnaire assessing seafood consumption at 32 w gestation. Consumption of seafood, eicosapentaenoic acid (EPA), DHA, and MeHg were estimated based on typical consumption patterns. Groups reporting no seafood consumption to some (up to 340 g/w) or exceeding 340 g/w were compared in multivariate logistic regression models including 29 variables. Outcomes were assessed among children (n=8,946) at 6, 18, 30 and 42 m (Denver Developmental Screening Test-DDST) and 8y (Strengths and Difficulties Questionnaire-SDQ) and the Wechsler Intelligence Scale for Children-III. Maternal depression was assessed by the Edinburgh Postnatal Scale of Depression (EPSD) at 18 w and 32 w gestation plus 8 w and 32 w postnatal. Findings. Maternal compliance (consuming <340 g/w) increased risks of their children scoring in the lowest quartile for verbal IQ (none: odds ratio 1.42, 95% confidence interval, 1.12-1.81, p<0.004) (some: odds ratio 1.09, 95% confidence interval, 0.92-1.29, ns) (overall model, p<0.02), compared to mothers exceeding the recommendation, after adjustment for potential confounding variables. Maternal compliance also increased risks for pathological scores in fine motor, communication and social subscales of the DDST and in peer problems and prosocial subscales of the SDQ. Risk of maternal depression increased during and after pregnancy. Conclusions. The toxicological risks and nutritional benefits were aggregated by evaluating seafood intake as a whole in a western population. Consumption of less than 340 g/w of seafood during pregnancy increased risks for pathological outcomes in the neurodevelopmental domains where protection was intended by the 2004 advisory. Limitation of fish consumption was also associated with increased risks for pathological prosocial and peer problems at 8 years of age and increased risk of maternal depression during and after pregnancy. This study stimulated the US Food and Drug Administration to conduct a scientific quantitative reevaluation modeling the potential topological risks from trace methyl-mercury and the nutritional benefits from seafood in the US population. This draft report, which modeled all relevant available published data, confirmed the findings of this study. This FDA evaluation may constitute the scientific basis for a revision of the 2004 advisory.

2. Development of high throughput quantitative assessments of omega-3 fatty acid status. A major impediment to the translation of the assessment of omega-3 fatty acid deficiencies on population scale has been the lack of low cost high throughput analytically accurate methods to assess omega-3 fatty acid status. We have developed a robotic high throughput methodology to quantitatively assess essential fatty acids in human plasma in low volumes at low cost. We are currently applying this method among approximately 9,000 mother infant pairs who are enrolled in the ALSPAC cohort to assess their long chain omega-3 status at birth and it nine years of age. These data will be used to assess neuropsychiatric and neurodevelopmental endpoints. In addition we have developed an active collaboration with the Center for Disease Control and developed methods for the quantitative analysis of trace metals including methyl-mercury, selenium, iron, cadmium and zinc in this well-characterized population (Robert Jones, PhD CDC). The measures will be utilized in risk benefit models to predict neurodevelopmental outcomes. This project has been co-funded by the National Oceanic and Atmospheric Administration (NOAA).

3. Metabolic studies on alcohol in long chain omega-3 turnover.
An intractable and long-standing problem in the field of EFA biology has been the relative efficacy of interconversion of 18- and 20-carbon precursors to longer chain, more unsaturated products. This problem can be addressed by dosing an animal with multiple stable isotope labels within a given pathway. These isotopes must be chosen so that they are distinguishable isotopomers when metabolized. We have been able to simultaneously give oral doses of two n-3 precursors and two n-6 precursors simultaneously and measure the conversion of each isotope independently to AA or DHA. This novel technique has been named MultiplE Simultaneous Stable Isotopes (MESSI). Our results in rats and human infants indicate that the 20-carbon intermediates are more efficiently converted to DHA than the 18-carbon intermediates when expressed on a per dose basis.
Over the past two decades the Section of Nutritional Neurochemistry has established that chronic alcohol use lowers brain composition of docosahexaenoic acid (DHA) in a series of clinical and animal models. Metabolic studies were conducted in the section utilizing deuterium labeled precursors. These were the first clinical studies performed in human adults assessing the effects of diets low in preformed EPA and DHA on the increased formation of EPA and DHA from precursors (linoleic acid and linolenic acid), their transportation and the effects of smoking and chronic alcohol use on this metabolism. Nonalcoholic smokers have an increased degradation of DHA and exhibit an increased compensatory production of DHA from precursors. Similarly, alcoholics exhibit increased degradation of DHA, and compensatory production. However, when the intensity of the alcohol challenge is too great, supplementation of DHA in the form of tablets cannot keep pace with its increase catabolism and tissue levels fall, consistent with our findings with chronic alcohol exposure in various experimental models. This fall in tissue levels of long chain polyunsaturated fatty acids then has adverse consequences to organ function, in particular hepatic steatosis and risk of fibrosis and brain apoptosis. Increasing the dietary intake of omega- 3 fatty acids in an alcoholic population may be protective for alcohol induced organ pathology. This variable may help to explain why some alcoholics develop hepatic steatosis and fibrosis and others don't. The alcohol-induced decline in brain DHA levels may contribute to the increased risk of major depression and aggressive disorders among alcoholics.

4. Long chain omega-3 fatty acid supplementation of alcoholics.
This randomized placebo-controlled intervention trial among aggressive alcoholics was designed to determine if supplementation in early abstinence reduced aggressive behaviors, reduced depressive symptoms, and altered cerebral spinal fluid levels of neurotransmitter metabolites. Subjects were randomized to receive either 2 g per day of EPA plus DHA or placebo oil. Subjects were followed as outpatients for 12 weeks monitoring relapse to alcohol and substance use and changes in craving, mood and aggression. 165 lumbar punctures were performed to obtain baseline and final measures on each subject. Unfortunately compliance by subjects was poor and the projected changes in tissue composition of omega-3 fatty acids were achieved in only 25% of the active agent randomized group. Thus, we were not able to evaluate efficacy in reducing relapse. However, in the study population as a whole, low CSF DHA content was correlated with smaller brain volume at baseline, indicating that low DHA status increased vulnerability to the adverse effects of alcohol on brain development. Lower CSF DHA content was also correlated with lower levels of metabolites of serotonin and dopamine in the CSF.

5. Suicide and inadequate long chain omega-3 fatty acid status
Epidemiologic data indicate that low fish consumption is a risk factor, but certainly not a sole determinant for suicide mortality. We examined 1,767 subjects in northern Finland and reported that frequent fish consumption (twice per week or more) significantly reduced the risk of reporting depressive symptoms (odds ratio = 0.63, p<0.03) and of reporting suicidal thinking (odds ratio = 0.57, p<0.04) after adjustment for confounding variables (Tanskanen et al., 2000). Lower fish consumption was associated with a doubling of the risk of suicidal thinking by mothers during pregnancy, after control for 29 confounding variables in a longitudinal cohort study enrolling 14,541 pregnancies (Golding, et al, in press 2009). These epidemiological observations are consistent with the assessment of omega-3 long chain fatty acid (LCFA) body compositions directly among patients. Among suicide att empters without depression as a primary diagnosis, low concentrations of plasma EPA alone were robustly correlated with greater psychopathology on rating scales of impulsivity, guilt, future suicide risk, and most subscales of the Comprehensive Psychopathological Rating Scale (Hibbeln et al., 2000). Low DHA status predicted greater risk of a new suicide attempt in a follow up study of more than 800 days, 5% of subjects above the median split had new attempts compared to 50% having new attempts among those below the median split (Sublette et al 2006). Resting PET scans of these subjects quantifying regional glucose uptake showed that future suicide attempters had greater activity in the anterior cingulate and limbic forebrain, consistent with the suspected pathophysiology of severe depression and PTSD. Low DHA in plasma phospholipid robustly predicted this regional hyperactivity (r = - 0.86, p<0.0001) indicating that low DHA status may increase limbic system mediated anxiety (Sublette et al, 2008). In a randomized blinded placebo controlled trial, we have reported a 45 % reduction in suicidal thinking and a 30% reduction in depression among patients with recurrent self harm recruited from an emergency room (Hallahan et al. 2007). This intervention used 2 g/d of omega-3 LCFAs in a 12 week trial of n = 49 subjects. Subjects also reported a reduced perception of daily stresses and anxiety which is likely relevant to reduction of risk of the development of PTSD.

6. Deficiencies in long chain omega-3 fatty acids and major depressive symptoms. One long-term effort of the Section on Nutritional Neurochemistry has been to evaluate the efficacy of seafood consumption or long chain omega-3 fatty acids in the treatment of major depressive symptoms and their application in other psychiatric disorders. In 2006 the section was instrumental in developing treatment recommendations issued by the American Psychiatric Association (Freeman et al 2006). This statement included a meta-analysis of randomized placebo-controlled intervention trials and indicated that the treatment effect size of long chain omega-3 fatty acids was equal to or greater than second-generation antidepressants.

7. Docosahexaenoic acid metabolism assessed by Positron Emission Tomography (PET) Docosahexaenoic acid (DHA, 22:6n-3) is a critical constituent of brain, but its metabolism has not been measured in the human brain in vivo. In monkeys, using positron emission tomography (PET), we first showed that intravenously injected [1-11C]DHA mostly entered non-brain organs, with approximately 0.5% entering brain. Then, using PET and intravenous [1-11C]DHA in 14 healthy adult humans, we quantitatively imaged regional rates of incorporation (K*) of DHA. We also imaged cerebral blood flow (rCBF) using PET and intravenous [15O]water. Values of K* for DHA were higher in gray than white matter regions and correlated significantly with values of rCBF in 12 of 14 subjects despite evidence that rCBF does not directly influence K*. For the entire human brain, the net DHA incorporation rate Jin, the product of K* and the unesterified plasma DHA concentration, equaled 3.8 ± 1.7 mg/day. This net rate is equivalent to the net rate of DHA consumption by brain and, considering the reported amount of DHA in brain, indicates that the half-life of DHA in the human brain approximates two and a half years. Thus, PET with [1-11C] DHA can be used to quantify regional and global human brain DHA metabolism in relation to health and disease (Umhau et al. 2009, in press).

[4] J.R. Hibbeln, R.V. Gow, The potential for military diets to reduce depression, suicide, and impulsive aggression: a review of current evidence for omega-3 and omega-6 Fatty acids, Military medicine, 179 (2014) 117-128.http://www.ncbi.nlm.nih.gov/pubmed/25373095

[37] T.L. Blasbalg, J.R. Hibbeln, C.E. Ramsden, S.F. Majchrzak, R.R. Rawlings, Changes in consumption of omega-3 and omega-6 fatty acids in the United States during the 20th century, The American journal of clinical nutrition, 93 (2011) 950-962.http://www.ncbi.nlm.nih.gov/pubmed/21367944